Seroprevalence of bluetongue disease in sheep in west and northwest provinces of Iran.

The objective of this study was to describe the seroprevalence rates of bluetongue virus (BTV) in sheep in west and northwest provinces of Iran. Bluetongue virus, an economically important orbivirus of the Reoviridae family, causes a hemorrhagic disease mainly in sheep and occasionally in cattle and some species of deer. Bluetongue virus is transmitted between its mammalian hosts by certain species of biting midges (Culicoides spp.) and it can infect all ruminant species. Overall, 26 serotypes have been reported around the world. Due to its economic impact, bluetongue (BT) is an Office of International des Epizooties (OIE)-listed disease. A total of 756 sera samples collected during 2007-2008, were available. Sera were tested with competitive enzyme-linked immunosorbent assay (C-ELISA). The seroprevalence rate in sheep was 40.87%. The rate of positivity in sheep in west and northwest was 46.10% and 33.75%, respectively. The highest prevalence of antibodies in serum was in West Azerbaijan (64.86%), and lower was in Ardabil (23.77%).


Introduction
Bluetongue (BT) is an insect-borne viral disease to which all species of ruminants are susceptible. It occurs mostly during periods of high temperature and rainfall, and usually disappears with the first frost or severe cold weather. 1 Bluetongue virus (BTV), the causative agent of BT of ruminants, have now been identified on all continents except for Antarctica. 2,3 Bluetongue virus is transmitted between its ruminant hosts almost exclusively through the bites of the females of vector species of the Culicoides spp. biting midge. 4,5 Specifically, BTV exists in an extensive band that includes tropical, subtropical, and temperate regions of the world between latitudes of approximately 40˚ north and 35˚ south. Exceptions are regions of Asia and western north America, where BTV infection of ruminants occurs as far as 50˚ north and, recently, northern Europe. 6,7 However, the distribution of specific insect vectors and different BTV serotypes differ remarkably throughout the world, so specific vector exist with specific constellations of BTV serotypes and topotypes in relatively distinct global ecosystems. 2,8,9 Although BTV is an orbivirus, it can occasionally be transmitted via seminal fluid and across the placenta. 10 Diagnostic tests are a major component of the success in any surveillance system. Wide varieties of tests are capable of detecting BTV-specific antibodies. These include agar gel immune-diffusion (AGID), hemagglutination-inhibition (HI), complement fixation (CF) and enzyme-linked immunosorbent assay (ELISA) either blocking ELISA or competitive ELISA (C-ELISA) which are serogroup-specific and serum neutralization (SN) test which is serotype-specific. 11 Only AGID and C-ELISA are recommended as prescribed tests for international trade in the Office of International des Epizooties (OIE) Manual of Standards for Diagnostic Tests and Vaccines. 12 Reports on BT outbreaks in the second semester of 2008 in Iran, 13 sera-positive herds in Turkey, 6,14 BT infection in Saudi Arabia, and outbreaks in Oman, and the Palestinian Autonomous Territories are among the most recent incidents of the disease in the region. 15 There have been few recent published studies from the region. Thus, we designed a study to evaluate the prevalence and distribution of serum antibodies to BTV in sheep in this area of Iran (Fig. 1).

Materials and Methods
Animals. In this study, for detection of specific antibodies to BTV in sheep blood serum samples by C-ELISA, 756 sheep blood samples collected from likely seropositive area in the west and northwest of Iran has investigated between June 2007 and September 2008.
C-ELISA. Anti-BTV antibodies were detected in serum samples by group specific, C-ELISA kit (ID-Vet, Monpellier, France). The test based on competitive between test sera and an anti-VP7 for a VP7 antigen previously bound to the solid phase of ELISA plate.

Results
Seroprevalence of bluetongue in west and northwest was shown in Table 1. The results showed that the seropositive rate in sheep over the whole study area was 40.87%. Presence of anti-bluetongue antibodies was found highest in west. The highest prevalence of antibodies was in West Azerbaijan (64.86%), and lower (23.77%) was in Ardabil (Table 1).

Discussion
It is worth mentioning that the situation of diagnosis of this virus in neighboring countries and the Middle-East (except Turkey and Occupied Palestine) is not better than our country. In such country as Saudi Arabia, Syria, Yemen and Pakistan, the presence of virus has been documented only relying on serological tests. 6,7,16 According to recent studies, there is an evidence of occurrence of BT disease in tropical and subtropical countries (such as Iran). In such areas generally, the disease appears sub clinically and does not attract attention. In such circumstances, the presence of the virus often confirms via serological evidence. It should be mentioned that in such foci, in spite of unrevealed disease and manifestation, sometimes sudden incidence of acute forms of the disease sustain a loss. [17][18][19] In Iran, identification of BTV in suspected cattle and sheep based on clinical manifestations was performed. However, there are some limitations and problems. First, it should be considered that clinical expression of BTV regarding strain and virus intensity, cattle race and environmental condition varies from per acute to subclinical. Second, symptoms of disease in sheep can be mistaken with those of other viral much diseases and even some of the non-viral diseases. 19 The objectives of the used C-ELISA test were both to confirm the BTV infected status of sheep in suspicious holdings in west and northwest of Iran (Diagnostic test). Thus, its use has been more and more often abandoned and replaced by C-ELISA tests, which are rapid and easier to use, more sensitive and specific. 20 In this study, the prevalence rate of BT antibodies in sheep was 40.87%. In other countries, the prevalence was as follows: 21.40% in Kazakhstan, 6 29.59% in southeastern Turkey, 14 62.69% in India, 21 54.10% in Saudi Arabia, 22 and 48.70% in Pakistan. 16 Climatic factors play an important role in the occurrence of BTV infection in animals and influence the size of vector populations and periods of their seasonal activity. 23 An analysis of climatic data was used to model the potential distribution of Culicoides imicola in Europe, predicting that might have spread from Spain, Greece and Italy to some areas along the Croatian coast as well as to the coastal areas of Albania, Serbia, Montenegro, Bosnia and Herzegovina. [24][25][26][27] Although more than 1000 species of Culicoides spp. are known worldwide, relatively few of these species have been incriminated as vectors of BTV. 28 Culicoides from western Turkey in relation to bluetongue disease of sheep and cattle was reported. 29 Species of vector insects that transmit BTV differ amongst regions, and are especially poorly characterized in the portions of Asia that are devoid of C. imicola, the traditional African-Asian vector of BTV. 2,7,29 Animals entering the western border of Iraq to Iran can be a cause of high titers of anti-bodies against the bluetongue virus in west of Iran. The prevalence of BT antibodies in sheep in the northwest of Iran was 33.75%. Although BTV infection of sheep is clearly widespread in northwest of Iran, the specific virus serotypes and vector insects that occur within the region remain uncharacterized, as they are in adjacent countries such as Kazakhstan. 6 The highest prevalence of BTV in sheep was in West Azerbaijan (64.86%) and Ilam (42.65%).
The results show that BTV infection is present in livestock animals in province. Some similar studies have been carried out in different area of country that mostly reported prevalence in the similar study. For example, 76.44% in East Azerbaijan, 30 34.70% in West Azerbaijan, 7 33.33% in Kerman, 31 45.90% in Kurdistan, 32 53.37% in Isfahan. 33 Furthermore, BTV infection in sheep apparently is largely subclinical. 7 During the BTV epidemics in Europe in 2008, Williamson and colleagues 34 considered clinical signs for diagnosis of the disease. The results showed low specificity of this method. These researchers believe that sometimes clinical signs of BTV in sheep are mistaken with those of such diseases as foot and mouth disease (FMD), ovine rinderpest, contagious ecthyma, and hemonchosis. 35,36 Iran located in the southeast of Europe makes it an important potential source of BTV strains and serotypes that might incur into adjacent areas. 5,37 In conclusion, seroprevalence of BTV has been never before reported in many area of Iran (Ardabil and Ilam). As per our knowledge, this is the first study was evaluated the prevalence of antibodies to BTV in sheep in some provinces of Iran. The results showed that a high incidence rate of BT antibodies has been detected in sheep in Iran that indicate serological evidence of exposure to infection was widely distributed in some provinces. There are no restrictions on the movement of animals from one region to another within the country. Thus, outbreaks may also occur due to transportation of animals. Consequently, a well-defined control strategy for preventing and controlling the BTV may be based not only on vaccination plans and vector eradication but also restriction on the movement of animals from one region to another within the country. As a vaccination for BT is not implemented in Iran, a seropositive result indicates BT infection in domestic population. 4,31 Further researches on the isolation and identification of BT virus in sheep are encouraged.